Lactosylceramide β1,3-N-acetylglucosaminyltransferase is an enzyme having an activity to transfer N-acetylglucosamine via β1,3-linkage to a galactose residue present in the non-reducing terminal of lactosylceramide (Galβ1-4Glc-ceramide). Neolacto-series glycolipids, lacto-series glycolipids, ganglio-series glycolipids, globo-series glycolipids and isoglobo-series glycolipids are synthesized from the lactosylceramide (Galβ1-4Glc-ceramide), and lactosylceramide β1,3-N-acetylglucosaminyltransferase is a key enzyme of the synthesis of neolacto-series glycolipids and lacto-series glycolipids.
Ganglioside GM3 (NeuAcα2-3Galβ1-4Glc-ceramide) is synthesized when GM3 synthase acts upon lactosylceramide. AsialoGM2 (GalNAcβ1-4Galβ1-4Glc-ceramide) is synthesized when GM2 synthase acts upon lactosylceramide. Since many other gangliosides are synthesized from GM3 and asialoGM2, GM3 synthase and GM2 synthase can be regarded as key enzymes of the synthesis of ganglio-series glycolipids. On the other hand, when lactosylceramide α1,4-galactosyltransferase acts upon lactosylceramide, Galα1-4Galβ1-4Glc-ceramide is synthesized and then a series of globo-series glycolipids are synthesized. When lactosylceramide α-1,3-galactosyltransferase acts upon lactosylceramide, Galα1-3Galβ1-4Glc-ceramide is synthesized and then a series of isoglobo-series glycolipids are synthesized.
Accordingly, it can be said that lactosylceramide α1,4-galactosyltransferase and lactosylceramide α1,3-galactosyltransferase are key enzymes of the synthesis of globo-series glycolipids and isoglobo-series glycolipids, respectively. It is considered that synthesis of a specific glycolipid in a cell is controlled by the expression and expression level of the above key enzymes.
Neolacto-series glycolipid is a glycolipid having Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide backbone, and lacto-series glycolipid is a glycolipid having a Galβ1-3GlcNAcβ1-3Galβ1-4Glc-ceramide backbone. Examples of the neolacto-series glycolipid include paragloboside (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide), sialylparagloboside (NeuAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide), NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-ceramide and the like. Examples of the lacto-series glycolipid include Galβ1-3GlcNAcβ1-3Galβ1-4Glc-ceramide, NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-ceramide, NeuAcα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc-ceramide and the like.
It has been found that lacto- or neolacto-series glycolipids to which fucose and sialic acid are added are accumulated in large amounts in many human cancers (particularly colon cancer or gastric cancer) [Annu. Rev. Immunol., 2, 103 (1984), Chem. Phys. Lipids, 42, 209 (1986)]. As a result of the measurement of glycosyltransferase activity in colon cancer tissues and their peripheral normal tissues or various colon cancer cell lines, it has been found that the activity of lactosylceramide β1,3-N-acetylglucosaminyltransferase is increased in colon cancer tissues and various colon cancer cell lines [J. Biol. Chem., 262, 15649 (1987)]. This result suggests that increase of the lacto- or neolacto-series glycolipids in colon cancer is caused by the increased lactosylceramide β1,3-N-acetylglucosaminyltransferase activity.
When a human promyelocytic cell line, HL-60, is treated with dimethyl sulfoxide or retinoic acid, it differentiates into granulocyte cells. On the other hand, when HL-60 is treated with phorbol ester such as phorbol-12-myristate-13-acetate (PMA), it differentiates into monocyte/macrophage. While neolacto-series glycolipids (paragloboside and sialylparagloboside) increase and ganglioside GM3 decreases when it is differentiated into granulocyte cells, ganglioside GM3 increases and neolacto-series glycolipids decrease when it is differentiated into monocyte/macrophage. Also, when HL-60 is cultured by adding a neolacto-series glycolipid, it differentiates into granulocyte cells, and when HL-60 is cultured by adding ganglioside GM3, it differentiates into monocyte/macrophage. The results show that expression of a specific glycolipid is important in determining the induction and direction of the differentiation. When HL-60 is treated with retinoic acid, the GM3 synthase activity does not change but the lactosylceramide β1,3-N-acetylglucosaminyltransferase activity increases [J. Biol. Chem., 267, 23507 (1992)]. Thus, it is considered that, in the HL-60 treated with retinoic acid, increase of neolacto-series glycolipids and decrease of ganglioside GM3 are induced caused by the increased lactosylceramide β1,3-N-acetylglucosaminyltransferase activity, and it differentiates into granulocyte cells as the result. On the other hand, when HL-60 is treated with PMA, the GM3 synthase activity increases and the lactosylceramide β1,3-N-acetylglucosaminyltransferase activity decreases [J. Biol. Chem., 267, 23507 (1992)].
Accordingly, it is considered that, in the HL-60 treated with PMA, increase of ganglioside GM3 and decrease of neolacto-series glycolipids are caused by the increased GM3 synthase activity and the reduced lactosylceramide β1,3-N-acetylglucosaminyltransferase activity, and it differentiates into monocyte/macrophage as the result. It is considered that lactosylceramide β1,3-N-acetylglucosaminyltransferase and GM3 synthase are taking an important role in determining the induction and direction of the differentiation of promyelocyte.
It is known that leukocytes express different glycolipids depending on their types and differentiation stages. For example, mature myelogenous cell expresses only neutral neolacto-series glycolipid [Mol. Cell. Biochem., 47, 81 (1982), J. Biol. Chem., 260, 1067 (1985)]. On the other hand, mature lymphocyte expresses only globo-series glycolipid [Mol. Cell. Biochem., 47, 81 (1982)]. It is suggested based on an analysis using leukocyte cell lines that the above differences of glycolipids are due to difference in the lactosylceramide β1,3-N-acetylglucosaminyltransferase activity. It has been found that the lactosylceramide β1,3-N-acetylglucosaminyltransferase activity is detected in myelogenous cell lines such as K-562, KG-1 and HL-60, but this enzyme activity is not detected in lymphocyte cell lines such as Reh, CCRF-CEM, MOLT-4, Ramos and RPMI 8226 [Archives of Biochemistry and Biophysics, 303, 125 (1993)].
It is known that a glycolipid having 3-sulfoglucuronic acid on the non-reducing terminal of its sugar chain (e.g., SO43GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide) is expressed at a specific period of time and in a specific region during the differentiation of nerve system. It has been suggested that this glycolipid is concerned in the mutual recognition of nerve cells and migration of nerves [J. Biol. Chem., 273, 8508 (1998)]. Since expression of the 3-sulfoglucuronic acid-containing glycolipid (SO43GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide) in nerve cells is controlled by lactosylceramide β1,3-N-acetylglucosaminyltransferase, it is considered that mutual recognition and migration of nerve cells are controlled by the expression of lactosylceramide β1,3-N-acetylglucosaminyltransferase [J. Biol Chem., 273, 8508 (1998)]. Since 3-sulfoglucuronic acid is recognized also by monoclonal antibody HNK-1 for a marker of human NK cell, it is also called HNK-1 epitope. Thus, it is considered that the 3-sulfoglucuronic acid-containing glycolipid plays an important role in the function of NK cell.
A sugar chain having GlcNAcβ1-3Gal structure is present in sugar chains of neolacto- and lacto-series glycolipids and also in N-linked sugar chains and O-linked sugar chains of glycoproteins, and in oligonsaccharide. For example, lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc) and lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc), which exist in human milk, or various oligosaccharides having them as backbones can be cited as the oligosaccharides having GlcNAcβ1-3Gal structure [Acta Paediatrica, 82, 903 (1993)]. The GlcNAcβ1-3Gal structure is also an element constituting a poly-N-acetyllactosamine sugar chain. The poly-N-acetyllactosamine sugar chain is a sugar chain having structure in which N-acetyllactosamine is repeatedly bound via β1,3-linkage [(Galβ1-4GlcNAcβ1-3)n; n is 2 or more], which is present in N-linked sugar chains and O-linked sugar chains of glycoproteins and also present in glycolipid sugar chains and oligosaccharides. Whether or not lactosylceramide β1,3-N-acetylglucosaminyltransferase uses substrates other than lactosylceramide, such as paragloboside, N-linked sugar chains and O-linked sugar chains of glycoproteins or oligosaccharides, has not been found.
Up to date, the lactosylceramide β1,3-N-acetylglucosaminyltransferase activity has been detected in colon tissues, colon cancer tissues, colon cancer cell lines (Colo205, SW403 and the like) and myeloid cell lines (K-562, KG-1 and HL-60), but there are no reports on the high purity purification of lactosylceramide β1,3-N-acetylglucosaminyltransferase [J. Biol. Chem., 262, 15649 (1987), Archives of Biochemistry and Biophysics, 260, 461 (1988), Carbohydrate Research, 209, 261 (1991), Archives of Biochemistry and Biophysics, 303, 125 (1993)].
On the other hand, regarding enzymes having the activity to transfer N-acetylglucosamine via β1,3-linkage to the galactose residue present in the non-reducing terminal of sugar chains (hereinafter referred to as “Gal β1,3-N-acetylglucosaminyltransferase”), there are reports on their partial purification but it is not clear whether these enzymes use lactosylceramide as a substrate [J. Biol. Chem., 268, 27118 (1993), J. Biol. Chem., 267, 2994 (1992), J. Biol. Chem., 263, 12461 (1988), Jpn. J. Med. Sci. Biol., 42, 77 (1989)].
Regarding cloning of genes, genes of two types of Gal β1,3-N-acetylglucosaminyltransferases have so far been cloned [Proc. Natl. Acad. Sci. USA, 94, 14294-14299 (1997), Proc. Natl. Acad. Sci. USA, 96, 406-411 (1999)]. It has been shown that β3GnT as one of them uses paragloboside as its substrate in vitro, but its activity is weak when lactosylceramide is used as the substrate [Glycobiology, 9, 1123 (1999)]. Also, it has not been found whether β3GnT uses lactosylceramide and paragloboside as its substrates inside cells. In addition, the presence of the other Gal β1,3-N-acetylglucosaminyltransferase is not clear.
Since a large number of sugar chains having the GlcNAcβ1-3Gal structure are present, it seems highly possible that two or more Gal β1,3-N-acetylglucosaminyltransferases having different acceptor specificity and expression tissue are present and have respective different functions. Accordingly, it is considered that lactosylceramide β1,3-N-acetylglucosaminyltransferase can be identified by cloning a Gal β1,3-N-acetylglucosaminyltransferase which is different from the two Gal β1,3-N-acetylglucosaminyltransferases so far cloned, and examining its acceptor specificity.
As described above, it is known that lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc) and lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc) or various oligosaccharides having them as backbones are present in human milk [Acta Paediatrica, 82, 903 (1993)]. These oligosaccharides have the GlcNAcβ1-3Gal structure in common. It is considered that they have a function to prevent babies from infection with viruses and microorganisms and a function to neutralize toxins. Also, they have an activity to accelerate growth of Lactobacillus bifidus which is a beneficial enteric bacterium. On the other hand, kinds of oligosaccharide existing in the milk of animals such as cows and mice are few and mostly lactose, and the above oligosaccharides existing in human milk are hardly present therein.
It may be industrially markedly useful if the above oligosaccharides contained in human milk or a milk containing them can be produced efficiently. When the gene of a Gal β1,3-N-acetylglucosaminyltransferase involved in the synthesis of the above oligosaccharides contained in human milk can be obtained, it is possible to use it in the efficient synthesis of the above oligosaccharides, but the enzyme has not been found yet.
Among sugar chains having the GlcNAcβ1-3Gal structure, particularly poly-N-acetyllactosamine sugar chain is a backbone sugar chain of many functional sugar chains (selectin ligand sugar chains, receptor sugar chains for microorganisms and viruses, SSEA-1 sugar chains, cancer-related sugar chains and the like) and deeply related to embryogenesis, cell differentiation or diseases such as inflammation and cancer. The poly-N-acetyllactosamine sugar chain also plays an important role in the stabilization of glycoprotein.
Since there is a possibility that Gal β1,3-N-acetylglucosaminyltransferases involved in the synthesis of poly-N-acetyllactosamine sugar chain functioning in respective cases are different, there is a possibility that a Gal β1,3-N-acetylglucosaminyltransferase different from the two enzymes so far cloned exists. There is a possibility that lactosylceramide β1,3-N-acetylglucosaminyltransferase is related to the synthesis of poly-N-acetyllactosamine sugar chain by transferring N-acetyllactosamine to sugar chains having Galβ1-4Glc or Galβ1-4GlcNAc at the non-reducing terminal (e.g., paragloboside) in addition to lactosylceramide.
Synthesis, function and application of the poly-N-acetyllactosamine sugar chain are described below.
The poly-N-acetyllactosamine sugar chain is synthesized by the mutual actions of a GlcNAc β1,4-galactosyltransferase (an enzyme having an activity to transfer galactose via β1,4-linkage to the N-acetylglucosamine residue present in the non-reducing terminal of sugar chains) and Gal β1,3-N-acetylglucosaminyltransferase. Regarding GlcNAc β1,4-galactosyltransferase, genes of four enzymes (β4Gal-T1, β4Gal-T2, β4Gal-T3 and β4Gal-T4) have so far been cloned, and acceptor specificity of each enzyme has been analyzed [J. Biol. Chem., 272, 31979-31991 (1997), J. Biol. Chem., 273, 29331-29340 (1997)].
Saccharides such as fucose, sialic acid, N-acetylgalactosamine and galactose, a sulfate group and the like are added to linear or branched poly-N-acetyllactosamine sugar chains to thereby form various cell-specific or stage-specific sugar chains (functional sugar chains, blood group sugar chains, cancer-related sugar chains and the like) [Glycobiology Series, (1) to (6), edited by Akira Kobata, Senitiroh Hakomori and Yoshitaka Nagai, published by Kodansha (1993)].
It is known that poly-N-acetyllactosamine sugar chains having a sialyl Lewis x sugar chain [NeuAcα2-3Galβ1-4(fucα1-3)GlcNAc] at their terminal are present on granulocytes, monocytes or activated T cells, and it is considered that these sugar chains relate to the accumulation of the leukocytes into inflammatory regions by functioning as ligands of adhesion molecules, E-selectin and P-selectin [Glycobiology Series, (1) to (6), edited by Akira Kobata, Senitiroh Hakomori and Yoshitaka Nagai, published by Kodansha (1993)].
It is also known that poly-N-acetyllactosamine sugar chains having a sialyl Lewis x sugar chain and a sialyl Lewis a sugar chain [NeuAcα2-3Galβ1-3(fucα1-4)GlcNAc] at the terminal are present on cancer cells such as colon cancer, and it is suggested that the sugar chains are also involved in tumor metastasis by functioning as ligands of E-selectin and P-selectin [Glycobiology Series, (1) to (6), edited by Akira Kobata, Senitiroh Hakomori and Yoshitaka Nagai, published by Kodansha (1993)].
It is known that the structure of the poly-N-acetyllactosamine sugar chain changes during the process of embryogenesis, cell differentiation or malignant transformation of cells [Glycobiology Series, (1) to (6), edited by Akira Kobata, Senitiroh Hakomori and Yoshitaka Nagai, published by Kodansha (1993)]. While a linear poly-N-acetyllactosamine sugar chain is expressed on human fetal erythrocytes, a branched poly-N-acetyllactosamine sugar chain is expressed on adult erythrocytes [Glycobiology Series, (1) “Various World of Sugar Chains”, edited by Akira Kobata, Senitiroh Hakomori and Yoshitaka Nagai, published by Kodansha, 1993]. ABO blood type antigens are expressed at the termini of the poly-N-acetyllactosamine sugar chains on erythrocytes. When a blood type antigen is expressed at respective termini of branched poly-N-acetyllactosamine sugar chains, it becomes a multivalent antigen and its binding ability with antibodies for blood type sugar chains increases 103 times or more in comparison with a linear type antigen.
It is known that a series of sugar chain antigens are systemically expressed during the developing stage of mouse early embryo. SSEA-1 (stage specific embryonic antigen-1) is a Lewis x sugar chain [Galβ1-4(fucα1-3)GlcNAc] existing at the terminal of a poly-N-acetyllactosamine sugar chain, and expression of the antigen starts at the 8-cell stage, reaches its peak at the morula stage and gradually disappears after the blastocyst stage [Glycobiology Series, (3) “Glycobiology of Cell Society”, edited by Akira Kobata, Senitiroh Hakomori and Yoshitaka Nagai, published by Kodansha, 1993]. The morula stage corresponds to a shifting stage in which germ cells so far proliferated by repeating simple numerical increase by cell division shift for the first time to the stage of blastocyst having differentiated “shape”. Just before forming blastocyst, the morula cells closely assemble and cause cell compaction. When an oligosaccharide having the SSEA-1 is added, this cell compaction is inhibited and normal development thereafter is also inhibited [J. Exp. Med., 160, 1591 (1984)]. It is also known that adhesion of mouse teratocarcinoma is inhibited by an anti-SSEA-1 antibody [Glycobiology Series, (3) “Glycobiology of Cell Society”, edited by Akira Kobata, Senitiroh Hakomori and Yoshitaka Nagai, published by Kodansha, 1993]. The above findings show that the SSEA-1 plays an important role in the development of early embryo by acting as an adhesion molecule or a sugar chain signal.
It is known that poly-N-acetyllactosamine sugar chains are expressed in a large quantity in cancer cells in comparison with corresponding normal cells [J. Biol. Chem., 259, 10834 (1984), J. Biol. Chem., 261, 10772 (1986), J. Biol. Chem., 266, 1772 (1991), J. Biol. Chem., 267, 5700 (1992)]. It is known that when N-ras proto-oncogene is expressed in NIH3T3 cell, molecular weight of N-linked sugar chain on the cell surface is increased and the cell acquires infiltrating ability, and at the same time, the amount of poly-N-acetyllactosamine sugar chain in the N-linked sugar chain is increased and activities of β1,4-galactosyltransferase and β1,3-N-acetylglucosaminyltransferase which relates to the synthesis of poly-N-acetyllactosamine sugar chain are also increased [J. Biol. Chem., 266, 21674 (1991)].
Galectins are a group of lectins having affinity for β-galactoside, which relate to the adhesion and signal transduction of cells, and their relation to diseases such as cancer is also suggested [Trends in Glycoscience and Glycotechnology, 9, 9 (1997)]. To date, 10 types of galectins are known in mammals. It is known that among these, galectin-1 and galectin-3 bind to linear poly-N-acetyllactosamine sugar chains with high affinity, and it is considered that certain glycoproteins containing these sugar chains are ligands of these galectins [Trends in Glycoscience and Glycotechnology, 9, 9 (1997), Trends in Glycoscience and Glycotechnology, 9, 47 (1997)].
Poly-N-acetyllactosamine sugar chains having sialic acids added to their termini serve as receptors for Mycoplasma and microorganisms [Acta Paediatrica, 82, 903 (1993)].
Thus, poly-N-acetyllactosamine sugar chains form backbone sugar chains of many functional sugar chains (selectin ligand sugar chains, receptor sugar chains for microorganisms and viruses, SSEA-1 sugar chain, cancer-related sugar chains and the like) and blood type sugar chains, and play important roles in efficiently presenting the sugar chains.
It is expected that poly-N-acetyllactosamine sugar chains having sialyl Lewis x sugar chains will become a medicament having anti-inflammatory effect or tumor metastasis inhibitory effect, as a selectin antagonist.
It is known that an oligosaccharide in which multivalent (four) sialyl Lewis x sugar chains (tetrasaccharides) is linked to poly-N-acetyllactosamine sugar chains shows the activity as a selectin antagonist at a low concentration of 1/100 or less in comparison with non-multivalent sialyl Lewis x sugar chains (tetrasaccharides) [J. Exp. Med., 182, 1133 (1995), Glycobiology, 6, 65 (1996), Glycobiology, 1, 453 (1997), Eur. J. Immunol., 27, 1360 (1997)]. Although a partially purified β1,3-N-acetylglucosaminyltransferase has been used for the synthesis of the poly-N-acetyllactosamine sugar chain moiety of the oligosaccharides, supply of this enzyme is a limiting factor so that it is difficult to synthesize a large amount of poly-N-acetyllactosamine sugar chains [Glycobiology, 7, 453 (1997)].
On the other hand, it is possible to synthesize poly-N-acetyllactosamine sugar chains by chemical synthesis, but its synthesis requires markedly complex steps [Tetrahedron Letter, 24, 5223 (1997)].
Accordingly, an efficient method for synthesizing poly-N-acetyllactosamine sugar chains is expected. Although the two types of Gal β1,3-N-acetylglucosaminyltransferases and their genes so far cloned may be used, it is considered that the use of other Gal β1,3-N-acetylglucosaminyltransferase having different substrate specificity and functions (e.g., lactosylceramide β1,3-N-acetylglucosaminyltransferase) and its gene may be efficient in some cases depending on the purpose.
The poly-N-acetyllactosamine sugar chains are also important for the stabilization of glycoprotein. Lysosome associated membrane glycoprotein-1 (lamp-1) and lysosome associated membrane glycoprotein-2 (lamp-2) are glycoproteins which exist in lysosome (partly exist on the cell surface) and almost completely cover inner face of lysosome membrane. Many sugar chains (some of them containing a poly-N-acetyllactosamine sugar chain) are added to lamp-1 and lamp-2 to prevent degradation of lamp-1 and lamp-2 by hydrolases in lysosome. It is known that when a human promyelocyte cell line HL-60 is treated with dimethyl sulfoxide, it differentiates into granulocyte cells, and during this differentiation process, the number of poly-N-acetyllactosamine sugar chains added to lamp-1 and lamp-2 increases and the metabolic rate (degradation rate) of lamp-1 and lamp-2 decreases [J. Biol. Chem., 265, 20476 (1990)].
Examples for increase of the ability to synthesize poly-N-acetyllactosamine sugar chains are shown below.
It is shown that poly-N-acetyllactosamine sugar chains are added to sugar chains of cell membrane glycoproteins when F9 cell is treated with retinoic acid or when Swiss 3T3 cell is treated with TGF-β [J. Biol. Chem., 268, 1242 (1993), Biochim. Biophys. Acta., 1221, 330 (1994)].
It is known that activities of β1,4-galactosyltransferase and β1,3-N-acetylglucosaminyltransferase involved in the synthesis of poly-N-acetyllactosamine sugar chains are increased, and the amount of poly-N-acetyllactosamine sugar chains in N-binding type sugar chains of glycoprotein is increased, when N-ras proto-oncogene is expressed in NIH3 T3 cells [J. Biol. Chem., 2, 21674 (1991)]. The molecular weight of a cell surface membrane protein CD43, CD45 or CD44 is increased when a core 2 β1,6-N-acetylglucosaminyltransferase gene is expressed in a T-cell line EL-4 [J. Biol. Chem., 271, 18732 (1996)]. The reason for this may be that sugar chains synthesized by the core 2 β1,6-N-acetylglucosaminyltransferase become a good substrate of β1,3-N-acetylglucosaminyltransferase involved in the synthesis of poly-N-acetyllactosamine sugar chains.
Also, it is known that the amount of poly-N-acetyllactosamine sugar chains added to lamp-1 or lamp-2 is increased when HL-60 cells are cultured at 27° C. [J. Biol. Chem., 266, 23185 (1991)].
However, there are no reports to date on the efficient production of recombinant glycoproteins to which poly-N-acetyllactosamine sugar chains are added, in host cells suitable for the production of recombinant glycoproteins (e.g., Namalwa cell, Namalwa KJM-1 cell, CHO cell). Accordingly, development of a process for efficiently producing a recombinant glycoprotein to which poly-N-acetyllactosamine sugar chains are added is an industrially important subject.
Although the two types of Gal β1,3-N-acetylglucosaminyltransferases so far cloned can be used, it is considered that use of other Gal β1,3-N-acetylglucosaminyltransferase having different substrate specificity and functions (e.g., lactosylceramide β1,3-N-acetylglucosaminyltransferase) may be efficient in some cases depending on the purpose.